Breast cancer remains the second leading cause of cancer related death in women. While the majority of these deaths are the result of metastatic spread, our understanding of the stages of metastasis and the operant features that drive secondary, tertiary, etc. tumors is sorely lacking. Given the known contributions of the tumor microenvironment to metastatic progression, identifying mechanisms of tumor-stromal interactions that contribute to extravasation and invasion will lead to novel therapeutic approaches that effectively target metastatic lesions. Among the most important players in the tumor microenvironment, myeloid-derived cells including macrophages, play a significant role in the priming of pre-metastatic sites, promoting the survival, proliferation, and invasion of tumor cells, enhancing immunosuppression, and fostering the development of therapeutic resistance. However, they can also contribute to the elimination of tumor cells, in part through the promotion of anti-tumor adaptive immune responses. In fact, there exist numerous macrophage populations in the metastatic niche with unique functions that may be pro-metasatic, tumoricidal, or both under different contexts. Our overarching goal in this proposal is to functionally profile the contributions of unique macrophage populations to metastatic seeding in the lung and elucidate the mechanistic basis for pro- and anti-tumorigenic functions of these cells. In Aim 1, we will quantify the function of unique, lung-resident macrophage subpopulations as drivers of metastatic extravasation and invasion. We have developed the capability to sort macrophage subpopulations from the lung using signatures of cell surface markers, allowing us to study individual phenotypes. Additionally, we have developed an in vitro microphysiologic model of metastatic extravasation and invasion. We will combine these tools to profile the pro-metastatic function of macrophage subpopulations from the lung by quantifying their ability to promote extravasation and invasion of 3 genomically unique breast carcinoma lines. In Aim 2, we will identify the molecular drivers of tumor cell-macrophage interactions that promote metastatic extravasation and invasion. Initial studies will be performed to transcriptionally profile distinct macrophage populations isolated from lungs bearing metastatic tumors, which will allow us to identify macrophage-derived factors that act on tumor cells and/or endothelial cells to promote extravasation. The results of these profiling studies will lead to the development of hypotheses that will be functionally assessed using the microfluidic model. As proof-of-concept, we will test STAT5 as a potential modulator of macrophage function in response to tumor cell-derived factors. Ultimately, we believe that delineating the specific signaling pathways within these cell types that control how they respond to tumor- derived cues and cancer-targeted therapies will allow us to direct their responses toward effective tumoricidal functions.